Device for carrying out lateral-flow assays involving more than one analyte

ABSTRACT

Disclosed is an improvement to a dry assay device for determining the concentration of a first analyte in a sample of body fluid and a second analyte in the same sample of body fluid. The device involves the use of a strip of an absorbent material through which the sample of body fluid flows and wherein the first analyte is determined calorimetrically in a first region of the strip and the second analyte is determined by an immunoassay which takes place in a second region of the strip located downstream from the first region. The improvement involves placing the strip in a hollow casing having a top and a bottom and which is so constructed that when the top and bottom of the casing are mated there is formed a U shaped, body fluid impervious barrier around the first region of the strip to prevent the sample of body fluid from flowing in any direction other than in the direction of the second region of the strip.

BACKGROUND OF THE INVENTION

[0001] Immunochromatographic strip formats have become increasinglypopular for qualitative and semi-quantitative assays which use visualdetection schemes. This type of assay involves the application of aliquid test sample suspected of containing the analyte to be detected toan application zone of an immunochromatographic test strip. The strip iscomprised of a matrix material through which the test fluid and analytesuspended or dissolved therein can flow by capillarity from theapplication zone to a capture zone where a detectable signal, or theabsence of such, reveals the presence of the analyte. Typically, thestrip will include means for immunospecifically binding the analyte tobe detected with its specific binding partner which bears the detectablelabel. In one such scheme, the strip contains an enzyme labeled, mobilebinding partner for the analyte which is in a zone downstream from thesample application zone. If analyte is present in the test sample, itwill combine with its labeled binding partner to form a complex whichwill flow along the strip to a detection zone which contains a substratefor the enzyme label which is capable of providing a colored response inthe presence of the enzyme. The strip may contain a zone in whichanalyte is immobilized, so that labeled binding partner which does notcombine with analyte, due to the absence of analyte in the sample, willbe captured and thereby inhibited from reaching the detection zone.There have been published various modifications of this technique, allof which involve some competitive specific binding system in which thepresence or absence of analyte in the test sample is determined by thedetection or lack thereof of labeled binding partner in the capturezone.

[0002] An alternative to the above described immunometric assay whichdetects the free labeled antibody is the so called sandwich format inwhich the capture zone contains immobilized antibodies against anepitope of the analyte which is different than the epitope to which thelabeled antibody is specific. In this format, there is formed a sandwichof the analyte between the immobilized and labeled antibodies and it istherefore an immunometric assay which detects the bound labeled antibodyspecies.

[0003] Not all of the schemes for immunochromatography rely on an enzymelabeled binding partner/enzyme substrate for providing the signal fordetection of the analyte. In U.S. Pat. No. 4,806,311 there is discloseda multizone test device for the specific binding assay determination ofan analyte and an immobilized binding partner therefore together with acapture zone for receiving labeled reagent which migrates thereto fromthe reagent zone. The capture zone contains an immobilized form of abinding substance for the labeled reagent. The labeled reagent bears achemical group having a detectable physical property which is detectableon the basis of such physical property, so that it does not require achemical reaction with another substance in order to be detected.Exemplary of such groups are colored species of fluorescers,phosphorescent molecules, radioisotopes and electroactive moieties.

[0004] U.S. Pat. No. 4,703,017 describes the use of visible particulatelabels for the receptor. Various particulate labels such as gold solparticles and visible dye containing liposomes are mentioned. InWO-96/34271 there is disclosed a device for determining a target analyteand creatinine in a fluid test sample which device has an assay stripfor the detection of creatinine and a second assay strip for thedetection of the target analyte. The creatine concentration can bedetermined calorimetrically or by the specific capture of labeledcreatinine binding partners. The concentration of the target analyte iscorrected based on the sample's creatinine concentration whichcorrection can either be done manually or by means of a properlyprogrammed reflectance analyzer.

[0005] EP 0 462 376 discloses an immunochromatographic procedure inwhich signal at the capture site and the conjugate recovery site of thestrip are detected and the analyte concentration is determined by theintensity of the signal at the capture site relative to the signal atthe conjugate recovery site.

[0006] Immunochromatographic strip formats provide a viable system forthe determination of various analytes (whether they be antigens orantibodies) but suffer from the limitation that they yield results whichare at best semi-quantitative when, for some analytes, more precise,quantitative results are required.

[0007] In WO-96/38720 there is disclosed a chromatographic assay devicefor the detection and/or determination of an analyte while giving apositive indication that flow has occurred properly through the device.The device comprises an opposable component including a samplepreparation zone and an absorber together with a second opposablecomponent including a first chromatographic medium withcapture/detection zones and a second chromatographic medium with acomparison zone and a comparison label zone. The opposable componentsare typically joined by a hinge so that the opposable components can befolded over upon each other to form a unitary cassette in which thechromatographic medium is encased.

SUMMARY OF THE INVENTION

[0008] The present invention is an improvement to a dry assay device fordetermining the concentration of a first analyte in a sample of bodyfluid and a second analyte in the same sample of body fluid. The firstanalyte is determined calorimetrically by the color change in a firstzone of a strip of absorbent material through which the body fluidsample flows and the concentration of the second analyte is determinedby an immunoassay in which the body fluid and analyte flow through asecond zone of the strip which is in fluid communication with the firstzone and analyte in the body fluid is immobilized in one of these zonesby interaction between the analyte and an immobilized specific bindingpartner to provide a detectable signal. The improvement comprisesplacing the strip of absorbent material in a hollow casing having a topand a bottom and which is constructed in a manner such that when the topand bottom portions of the casing are mated there is formed a U shapedbody fluid impervious barrier around the first zone of the strip therebypreventing the sample of body fluid from flowing in any direction otherthan towards the second zone of the strip.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 represents the strip component of the device of the presentinvention.

[0010]FIG. 2 is a top view of the casing portion of the present device.

[0011]FIG. 3 depicts the top and bottom of the casing segment of thedevice showing these segments before they are folded over each other andsnapped into place to house the strip.

DESCRIPTION OF THE INVENTION

[0012] Referring to FIG. 1, the strip 10 has a solid support 1 whichsupports a chemistry reagent pad 3 in which there are absorbed reagentsfor the colorimetric determination of a first analyte. Downstream fromthe chemistry reagent pad is the region 5 in which the immunoassay iscarried out. This region contains wicking pad 7 and reagent zone 9 whichcontains labeled antibodies specific for the analyte whose concentrationis being determined. These portions of the strip are depicted asoverlapping the next adjacent portion of the strip. This is an optionalconfiguration which provides for greater contact area between the zonesthus facilitating fluid flow through the strip. This is not essentialsince simple connectors such as head to tail contact are sufficient whenthe test fluid is one which can flow easily through the strip. The labelis preferably a visible particulate label such as gold sol, however, anenzymatic label could be used provided the capture zones 11 and(optional second capture zone 13) contain an appropriate substrate forthe enzyme. Capture zones 11 and 13 contain either immobilized analyteor an immobilized antibody specific for an epitope of the analytedistinct from that to which the labeled antibody is specific. In thefirst embodiment there takes place a competitive reaction in whichanalyte in the test fluid and that which is immobilized in the capturezone(s) compete for labeled antibody. In this format, the strength ofthe signal from the capture zone will be inversely proportional to theconcentration of analyte in the test fluid. In the sandwich format,there will be immobilized in the capture zone(s) antibodies specific toa second epitope on the analyte which is distinct from that to which thelabeled antibody is specific. In this format the strength of the signalfrom the capture zone(s) will be directly proportional to theconcentration of analyte in the test sample. The strip may also containa control region 15 which is typically a positive control in whichlabeled antibody is captured by a specific capture means such asimmobilized anti-mouse IgG. The strip will also normally have anabsorbant pad 17 which absorbs test fluid and thereby encourages itsflow through the strip.

[0013]FIG. 2 is a top view of the casing 20 which contains the strip ofFIG. 1. The casing has an application port 21 through which the testsample is applied and any color change in the chemistry reagent pad 3can be observed. There is a second viewing port 23 through which thecapture band(s) 11 and 13 as well as the optional collection band areviewed.

[0014] The top of the casing may be provided with a second viewing port25 through which other colored indications may be viewed. For example,the strip may contain colored bands which are coded to identify theassay or a thermochromic liquid crystal which can be used to measure thetemperature of the strip, so that corrections based on temperaturerelated variables can be made.

[0015] The directional flow features of the present invention areillustrated by FIG. 3, in which the casing 30 is shown with its top 31and bottom 33 in the open position. The top of the casing has anindentation 35 and the bottom of the casing has an indentation 37 whichform a hollow chamber of sufficient size to hold the strip when the topand bottom are mated after placing the strip in the indentation 37 inthe casing's bottom portion. The casing is designed to hold the strip sothat the chemistry reagent pad 3 is aligned with the sample applicationport and surrounded on three sides by the U shaped barrier 39 which,when the top and bottom of the casing are mated, forms a fluidimpervious barrier around this region of the strip. The U shaped barrierprecursors 39 a and 39 b which form the barrier 39 by contact betweenbarrier precursors 39 a on the top portion of the casing and 39 b on thebottom portion when the top and bottom are mated need not be equallydivided between the top and bottom of the casing. The only requirementis that precursor 39 a on the casing top and 39 b on its bottom joinsnugly when the casing top and bottom are mated to thereby form a Ushaped dam that prevents the fluid sample from flowing in any directionother than towards the second and subsequent zone(s) of the strip inwhich the immunoassay is carried out. While barrier precursors 39 a and39 b will both normally project above the plane of the casing's top andbottom respectively, this is not critical since barrier precursor 39 aor 39 b can be in the same plane as the casing top or bottom with all ofthe projecting portion necessary to form barrier 39 being on the othersurface. As represented by the phantom portions of FIG. 3, 40a and 40 b,the barrier can extend further down the strip to extend beyond thewicking portion of the immunochromatography portion. This will provideadditional reliability by insuring that fluid sample entering the sampleport 21 will fully inoculate the immunoassay portion of the strip.

[0016] In another embodiment of the invention, barrier precursor 39 a isslightly narrower than chemistry reagent pad 3 and precursor 39 b ismolded to have a height from the casing bottom's surface less than thethickness of the pad, so that when the casing's top and bottom are matedthe chemistry reagent pad is squeezed between barrier precursors 39 aand 39 b to reduce the chance that a capillary gap will form between thewalls of the casing and the reagent pad. If such a gap were to form, thefluid sample introduced through the entry port would travel into the gapand not toward the wick of the immunoassay portion of the strip. Thebottom portion of the casing can be equipped with troughs 41 on one orboth sides of the area in which the strip rests to serve as drainagefields which serve to remove any excess fluid sample applied to thesample port. The casing bottom can be advantageously equipped with aridge 43 which is in the outline of the strip and serves to ensureproper placement in the casing. This ridge should be fairly shallow, sothat excess test fluid can flow over it into drainage troughs 41. Thecasing top and bottom can also be equipped with a series of pins 45which lock up with holes 47 when the top and bottom of the casing aremated to hold them snugly together. The cassette top may be equippedwith pressure bars 48 which are designed to hold the strip firmlyagainst the bottom of the casing when the casing top and bottom aremated to prevent fluid sample from flowing under the strip. Depression49 in the bottom of the strip is optionally present to hold a desiccantbead.

[0017] The strip can be prepared from any matrix material through whichthe test fluid carrying the analyte, labeled binder and/or labeledbinder-analyte conjugate contained therein can flow by capillarity andcan be of a material which is capable of supporting non-bibulous lateralflow as described in U.S. Pat. No. 4,943,522 as liquid flow in which allof the dissolved or dispersed components of the liquid are carriedthrough the matrix at substantially equal rates and with relativelyunimpaired flow as contrasted to preferential retention of one or morecomponents as would be the case if the matrix material were capable ofabsorbing or imbibing one or more of the components. An example of suchmatrix material is the high density or ultra high molecular weightpolyethylene sheet material from Porex Technologies. Equally suitablefor use as the matrix from which the chromatographic strip can befabricated are bibulous materials such as paper, nitrocellulose andnylon.

[0018] Various immunochromatographic strip formats are suitable forthose portions of the strip which are downstream from the pad containingthe colorimetric reagents. The type of chemistry reagent pad may varydepending on the analyte of interest relative to the immunoassay. Thereagent pad generally consists of an absorbent material such as a paperor membrane that has been impregnated with a respective reagentassociated with a particular test to be performed. With urinalysistesting, this reagent pad may be, for example, a test for creatinine, atest for leukocytes, a test of pH or a test of blood. An adhesivebacking is placed on the dried, impregnated paper and cut into a ribbonof a desired width. The ribbon is adhered to a support at a locationthat would place the reagent under the sample port of the casing. Onceall the immunoassay components are in place on the support, the supportis cut to the dimensions that are needed for the strip to lie in thecavity of the casing bottom. When the reagent pad encounters the sample,the pad changes color over time and the reflectance of the color, whichis proportional to the amount of analyte present in the sample, ismeasured. A particularly suitable format is that which is disclosed inU.S. Pat. No. 4,446,232 in which there is described a device for thedetermination of the presence of antigens, which device comprises astrip of matrix material having a first zone in which there are providedimmobilized analyte and enzyme linked antibodies specific to the analyteto be determined. The labeled antibodies can flow to a second zone whenreacted with analyte introduced into the first zone via the test samplebut will not so flow in the absence of analyte in the test fluid sincethe labeled antibodies will be bound in the first region by interactionwith the immobilized analyte. The analyte is typically an antigen,although the format can be designed to detect the presence of antibodiesas analyte. An alternative to this format is a sandwich format in whichthe labeled antibody is specific for one epitope of the analyte andthere is immobilized in the capture zone a second antibody which isspecific to a second epitope of the analyte so that there is formed inthe capture zone an antibody-analyte-labeled antibody sandwich in thepresence of analyte in the fluid test sample. As an alternative to theuse of an enzyme label, the antibodies used in the device can be labeledwith a visible particulate label such as colored latex or metal sol.This is the preferred form of labeling, although any physicallydetectable signal generator may be used as the label.

[0019] In operation, the device is used by pipetting the fluid sample,which is typically urine, through the sample application port 21. Thiswill result in wetting of the pad containing the colorimetric reagentsand a reaction between the first (reference) analyte and thecolorimetric reagents for the determination of this analyte. Suchreagents can comprise an oxidase enzyme, a pseudoperoxidase and anoxidizable dye so that interaction between the reagent system andanalyte in the test fluid will produce a colored response upon oxidationof the dye. A common reference analyte in urinalysis is creatinine, theend metabolite when creatine becomes creatine phosphate which is used asan energy source for muscle contraction. The creatinine produced isfiltered by the kidney glomeruli and then excreted into the urinewithout reabsorption. In order to increase the sensitivity of urinaryassays and minimize the problem of high urine flow rates which result inurine dilution, analyte/creatinine ratios are used in urine analyteassays to normalize the urine concentration. Common creatinine assaysinclude the alkaline Jaffe and Benedict-Behre methods which are run at ahigh pH, typically in the range of from 11.5 to 12.5. More recently,there has been developed a creatinine assay in which the urine sample iscontacted with cupric ions in the presence of citrate, a hydroperoxideand an oxidizable dye which provide a colored response in the presenceof oxygen free radicals and a pseudoperoxide. This method is more fullydescribed in U.S. Pat. No. 5,374,561 incorporated herein by reference.Referring to FIG. 1, the present invention can be used for thedetermination of protein in urine by incorporating the creatininereagent into colorimetric chemistry reagent pad 3. Upon application ofthe urine test sample the creatinine concentration can be determinedcalorimetrically such as by the use of a reflectance spectrometer. Theurine sample will continue to flow down the strip of absorbant material,through the wicking pad 7 and reagent zone 9. The U shaped barrier,which surrounds at least the chemistry reagent pad 3, prevents the testsample from flowing in any direction other than downstream from this padthereby improving the accuracy of the assay which is carried out usingthe test strip. Extending the legs of the U shaped barrier further downthe strip, to cause them to be co-extensive with the wicking pad 9 oreven further down the strip will further enhance the device's accuracy.After flowing through the wicking pad 9, and into reagent zone 9, thetest sample contacts the labeled antibodies which flow along with thefluid sample towards the capture zone 11 where the labeled antibodiesare captured either by interaction with immobilized analyte orinteraction between analyte in the fluid test sample, the labeledantibodies specific thereto and antibodies immobilized in the capturezone which are specific to another epitope on the analyte to form asandwich. Regardless of how the labeled antibodies are captured in thecapture zone, there will be generated two signals in the strip; thefirst by the interaction of creatinine in the urine test sample with thecreatinine reagent in reagent pad 3 and the second from the labeledantibody in capture zone 11. These signals can be read by a properlyprogrammed reflectance spectrometer and rationalized to give a resultwhich is the urine sample's protein concentration which has beencorrected for the urine's flow rate by using the creatinineconcentration.

[0020] The reference analyte is not limited to creatinine since anyreference analyte whose concentration in a sample of body fluid isclinically related to the concentration of the target analyte can bemeasured by its reaction with the reagent pad 3. Thus, for example, thebody fluid tested can be whole blood, the target analyte can be HbA_(1c)and the second analyte can be total hemoglobin since the apparentconcentration of HbA_(1c) can be adjusted to the whole blood's totalhemoglobin concentration to factor out bias in the HbA_(1c) assay.Inulin, administered intravenously, is, like creatinine, an indicator ofrenal flow. Clinically significant results can be obtained bydetermining the ratio of these pairs of analytes in the sample of bodyfluid.

[0021] Many clinically significant target analytes are present in urineand as determinable by means of the present invention. Among theseanalytes are deoxypyridinoline, human serum albumin, drugs of abuse suchas amphetamines/barbiturates/cocaine, clinically important proteinmarkers such as prostate specific antigen, kidney disease proteins suchas lactate dehydrogenate, N-acetyl-B-D-glucosamine, pregnancy orfertility associated hormones such as human chorionic gonadotropin andmarkers of urinary tract infection.

[0022] While the means for detecting the signal from the developed stripof the device of the present invention will depend on the detectablelabel attached to the labeled binding partner, the use of a reflectancespectrometer is typical when the label's detectable physical property isthe reflectance of light at a predetermined wavelength. In a preferredmethod of using the device there is provided a reflectance meter withmeans for moving the strip or the meter's detector element relative toeach other such as by use of a specimen table for the strip which can bemoved laterally under the readhead of the detector. The reflectance fromthe chemical reagent pad can be read to obtain the concentration of thisreference analyte in the fluid sample and then the device can be shiftedon the specimen table for reading the concentration of the targetanalyte to provide raw data which the reflectance spectrometer'spre-programmed software can use to provide the corrected concentrationof the target analyte.

[0023] The method of practicing the present invention is more fullyillustrated by the following example:

EXAMPLE I

[0024] A study was carried out testing the fluid sample flowcharacteristics within two different casing types; one with (1) and onewithout (2) the “U” shaped barrier. The strip design used in model 1 wasconstructed to incorporate the wicking pad of the immunoassay portion tolay beneath the creatinine reagent and was referred to as the underpadformat. The model 2 casing had both the underpad format and a stripformat wherein the wicking pad for the immunoassay came within 0.0245″of the creatinine reagent pad. The creatinine reagent pad demonstratedin this example was paired with a deoxypyridinoline (Dpd) immunoassay.The creatinine reagent pad was made of an absorbent paper impregnatedwith reagents to provide a test based on the peroxidase like activity ofa copper creatinine complex which catalyzes the reaction ofdiisopropylbenzene dihydroperoxide and 3, 3′, 5, 5′ tetramethylbenzidineto provide a color change in the presence of creatinine.

[0025] The study was analyzed for two effects, i.e. the number offailures when the Dpd immunoassay was not inoculated after adding thesample and for which casing format provided better performance (% Cv)for the Dpd capture bands using the reflectance value at 565 nm. A thirdstrip format (“dip and read”) was used as a control. A buffer solutioncontaining Dpd and creatinine concentrations within the intended rangewas used as control. Testing was done using 15 replicates for eachformat, except for model 2 (underpad) which used only 7 replicates. Theresults of the study were that (1) there were no failures of inoculationof the Dpd immunoassay for either casing model, although in a previousstudy there was noted a failure of inoculation of the model 1 cassettes;(2) the model 2 casing provided better performance in terms of lower %CV for the Dpd immunoassay as shown in the following table. %reflectance at 565 nm Format mean SD % CV Model 1 50.5 2.3 4.5 cassette(underpad) Model 2 49.4 1.7 3.5 cassette (underpad) Model 2 47.7 1.1 2.4cassette (0.025″ gap) “dip and read” strip 48.3 1.3 2.8

1. In a dry assay device for determining the concentration of a firstanalyte in a sample of a body fluid and a second analyte in the samesample of body fluid in which the first analyte is determinedcolorimetrically by the color change in a first zone of a strip ofabsorbent material through which the body fluid sample can flow and theconcentration of the second analyte is determined by an immunoassay inwhich the body fluid and analyte flow through a second zone of the stripwhich is in fluid communication with the first zone and analyte labeledspecific binding partner conjugate in the body fluid is immobilized inone of these zones by interaction between the analyte or the specificbinding partner and an immobilized binder in a separate zone or thestrip to provide a detectable signal, the improvement which comprisesplacing the strip in a hollow casing constructed of a body fluid sampleimpervious solid material having a top and a bottom which when matedprovide a hollow chamber suitable for holding the strip which chamber isin fluid communication with the exterior of the casing and when the topand bottom of the casing are mated there is formed a U shaped, fluidimpervious barrier around the first discrete zone of the strip whichprevents test fluid from flowing in any direction other than in thedirection of the second zone and any subsequent zone(s) of the strip. 2.The device of claim 1 wherein the concentration of the first analyte isclinically related to that of the second analyte.
 3. The device of claim1 wherein a portion of the U shaped barrier is fixed to the top of thecasing and a portion is affixed to the bottom of the casing.
 4. Thedevice of claim 1 wherein the U shaped barrier extends downward alongthe strip beyond the end of the first discrete zone.
 5. The device ofclaim 1 wherein the second zone of the strip is divided into sub zonesone of which is an absorbant wicking pad and the U shaped barrierextends downward along the strip to the end of this sub zone.
 6. Thedevice of claim 1 wherein the top and bottom portion are constructed sothat a press fit secures them together to form the casing having thehollow chamber.
 7. The device of claim 1 wherein the casing is made ofplastic.
 8. The device of claim 7 wherein the plastic is polystyrene, anacrylic polymer or a polyurethane.
 9. The device of claim 1 wherein thebottom portion of the casing is equipped with a ridge in the shape ofthe strip to ensure proper placement of the strip in the casing.
 10. Thedevice of claim 1 wherein the body fluid is urine.
 11. The device ofclaim 10 wherein the first analyte is creatinine and the second analyteis Dpd.
 12. A device for detecting the concentration of a target analytein a sample of body fluid relative to the concentration of a referenceanalyte in the same sample of body fluid which comprises: i. a strip ofabsorbant material through which the test sample can flow which stripcontains: a) a first zone which contains reagents for the colorimetricdetermination of the reference analyte; b) a second zone containing areleasable specific binding reagent for the target analyte whichspecific binding reagent bears a detectable label and forms ananalyte/labeled specific binding partner reagent upon contact with thebody fluid sample containing the target analyte; and c) a third regionwhich contains an immobilized capture reagent for specifically bindingthe analyte or labeled specific binding reagent for the analyte; and ii.a hollow casing having a top and bottom segment enclosing the strip ofabsorbent material which casing has a body fluid inlet port directlyabove and in visual and fluid communication with the first zone of thestrip of absorbent material and one or more view ports for detecting theamount of detectable label captured in the third region wherein thehollow casing has a U shaped body fluid impervious barrier whichsurrounds three sides of the first region of the strip thereby allowinga sample of body fluid applied to the first region of the strip throughthe inlet port to flow only in the direction of the second and thirdregions.
 13. The device of claim 12 wherein the top and bottom segmentare constructed so that a press fit secures them together to form thecasing.
 14. The device of claim 12 wherein the casing is made ofplastic.
 15. The device of claim 14 wherein the plastic is polystyrene,an acrylic polymer or a polyurethane.
 16. The device of claim 12 whereina portion of the U shaped barrier is embossed from the top segment ofthe casing and a portion is embossed from the bottom portion so thatwhen the top and bottom portions are mated around the strip there isformed the complete “U” shaped barrier.
 17. A method for determining theconcentration of a first analyte and a second analyte in a sample ofbody fluid which method comprises applying the sample of body fluid tothe strip of claim 12 and determining the response in the first zone andthe response in the second zone.
 18. The method of claim 17 wherein thebody fluid is urine.
 19. The method of claim 18 wherein the firstanalyte is creatinine and the second analyte is Dpd.
 20. The method ofclaim 17 wherein the concentration of the first analyte is clinicallyrelated to the concentration of the second analyte and the secondanalyte's observed concentration is corrected based on the observedconcentration of the first analyte.
 21. The method of claim 20 whereinthe body fluid is urine, the first analyte is creatinine and the secondanalyte is Dpd.